Journal: Nature Communications

Article Title: Structural basis for assembly and disassembly of the IGF/IGFBP/ALS ternary complex

doi: 10.1038/s41467-022-32214-2

Figure Lengend Snippet: a Fluorescence-detection size-exclusion chromatography (FSEC) of binary or ternary complexes after 10 h proteolysis by thrombin. The elution profile for standard IGF1-GFP (yellow), IGF1-GFP-strep/IGFBP3 binary complex (blue), and IGF1-GFP/IGFBP3/ALS-His ternary complex (red) are indicated as a control (top). b Immunoblot analysis of IGF1/myc-IGFBP3-Strep/ALS complex after 10 h proteolysis by thrombin and subsequent SEC. Anti-myc and anti-Strep antibody were used to detect NBP3 and CBP3, respectively. No-thrombin treatment served as negative control (Ctrl). c FSEC of the ternary complex (IGF1/IGFBP3-GFP/ALS-His) after thrombin digestion for indicated time. Fluorescence signal (GFP) was monitored to examine the dissociation of CBP3 from the ternary complex. d Quantification of immunoblot analysis for IGF1R phosphorylation after treatment of intermediate ternary complex. HEK293A cells were treated with IGF1-His (5 nM, positive control), IGF1-His/IGFBP3/ALS ternary complex (5 nM, none), or intermediate ternary complex (5 nM). The intermediate ternary complex was prepared by protease digestion (thrombin, ADAM12 and PAPP-A2) and subsequent SEC purification. No treatment was used for negative control (Ctrl). Data from three independent experiments ( n = 3) were analyzed and relative pIGF1R/IGF1R (Fold to Ctrl) were expressed as mean ± SD (ns, not significant vs. ctrl; **** P < 0.0001 vs. ctrl; #### P < 0.0001 vs. none). P -values by one-way ANOVA test followed by Sidak’s multiple comparisons test. e SDS-PAGE analysis of the IGF1/hybrid-BPs/ALS ternary complex after thrombin digestion. f Quantification of immunoblot analysis for IGF1R phosphorylation with intermediate ternary complex containing hybrid-BPs. IGF1-His (5 nM, positive control), IGF1-His/IGFBP3/ALS ternary complexes (5 nM), or IGF1-His/hybrid-BPs/ALS ternary complexes (5 nM) with and without thrombin digestion were treated onto HEK293A cells. No treatment for negative control (Ctrl). Data from five independent experiments ( n = 5) were analyzed and relative pIGF1R/IGF1R (Fold to Ctrl) were expressed as mean ± SD (ns, not significant; **** P < 0.0001 Ctrl vs. IGF1-His or -thrombin vs. +thrombin for each group). P -values by one-way ANOVA test followed by Sidak’s multiple comparisons test. g , h FSEC of the ternary complex IGF1-GFP/hybrid-BPs/ALS-His ( g ), or IGF1/hybrid-BPs-GFP/ALS-His ( h ) after thrombin digestion. Fluorescence signal (GFP) was monitored to examine the dissociation of IGF1 or CBP3 from the indicated ternary complex.

Article Snippet: IGF1R phosphorylation was assessed by first blocking the blot by incubation with 5% (w/v) skim milk in TBS-T at RT for 1 h, and then incubating it with an anti-phospho-IGF1R primary antibody (Y1135/1136) (1:1000 dilution, #3024, Cell Signaling) at 4 °C for 12 h, followed by incubation with HRP-conjugated goat anti-rabbit IgG antibody (1:5000 dilution, #31460, Thermo Fisher Scientific).

Techniques: Fluorescence, Size-exclusion Chromatography, Control, Western Blot, Negative Control, Phospho-proteomics, Positive Control, Purification, SDS Page

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: The Long Noncoding RNA 150Rik Promotes Mesangial Cell Proliferation via miR-451/IGF1R/p38 MAPK Signaling in Diabetic Nephropathy.

doi: 10.1159/000495590

Figure Lengend Snippet: Fig. 6. 150Rik regulated mesangial cell proliferation via miR-451/IGF1R/IGF1/p38MAPK signalling pathway. A, The expression of IGF1R was measured by Western blot when 150Rik was over-expressed and down-expressed. And the gray values of the bands were quantitative analyzed. B, The cell proliferation of MCs transfected with 150Rik (+), si150Rik, miR-451 mimics, miR-451 inhibitor,150Rik (+) +miR-451 inhibitor and si150Rik+ miR-451 mimics was measured by EdU when IGF1R was specifically inhibited by picropodophyllin (PPP). Scale bar, 200µm. C, The protein expressions of IGF1, p38MAPK and p-p38MAPK were measured by Western blot in MCs transfected with150Rik (+), si150Rik, miR-451 mimics, miR-451 inhibitor,150Rik (+) +miR-451 inhibitor and si150Rik+ miR-451 mimics. And the gray values of the bands were quantitative analyzed. The data were representative of the results three independent experiments, and the data were presented as means± SD. (**P<0.01, NS, no significant).

Article Snippet: The primary antibodies used in this study were: IGF1R (Proteintech, USA, 1:1000), cyclin D1 (Proteintech, USA, 1:1000), IGF1 (Sangon,China, 1:500), p38MAPK (Proteintech, USA, 1:500), p-p38MAPK (Proteintech, USA, 1:500) and GAPDH (Glyceraldehyde-3-phosphate Dehydrogenase) (Abcam, USA, 1:5000).

Techniques: Expressing, Western Blot, Transfection

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: The Long Noncoding RNA 150Rik Promotes Mesangial Cell Proliferation via miR-451/IGF1R/p38 MAPK Signaling in Diabetic Nephropathy.

doi: 10.1159/000495590

Figure Lengend Snippet: Fig. 7. 150Rik regulated mesangial cell proliferation via miR-451/IGF1R/IGF1/p38MAPK signaling pathway by immunofluorescent and Western blot. A, The protein expressions of IGF1, p38MAPK and p-p38MAPK were measured by immunofluorescent in MCs transfected with150Rik (+), si150Rik, miR-451 mimics, miR- 451 inhibitor, 150Rik (+) +miR-451 inhibitor and si150Rik+ miR-451 mimics. Scale bar, 200µm. B, The protein expressions of IGF1R and p-p38MAPK were measured by Western blot in MCs transfected with 150Rik (+), si150Rik, miR-451 mimics, miR-451 inhibitor, 150Rik (+) +miR-451 inhibitor and si150Rik+ miR-451 mimics when IGF1R was specifically inhibited by PPP. The data were representative of the results three independent experiments, and the data were presented as means±SD. (**P<0.01, NS, no significant).

Article Snippet: The primary antibodies used in this study were: IGF1R (Proteintech, USA, 1:1000), cyclin D1 (Proteintech, USA, 1:1000), IGF1 (Sangon,China, 1:500), p38MAPK (Proteintech, USA, 1:500), p-p38MAPK (Proteintech, USA, 1:500) and GAPDH (Glyceraldehyde-3-phosphate Dehydrogenase) (Abcam, USA, 1:5000).

Techniques: Western Blot, Transfection

Journal: Translational Oncology

Article Title: IGF1R Axis Inhibition Restores Dendritic Cell Antitumor Response in Ovarian Cancer

doi: 10.1016/j.tranon.2020.100790

Figure Lengend Snippet: Reduced total and phosphorylated IGF1R protein levels in differentiated DCs. (A) Human leukemic THP-1 cells were differentiated to immature DCs by treatment with 100 ng/ml IL-4 and 100 ng/ml GM-CSF for 5 days and then differentiated to DCs by treatment with 200 ng/ml IL-4, 100 ng/ml GM-CSF, 20 ng/ml TNFα, and 200 ng/ml Ionomycin for 2 days. THP1 scale bar = 50 μm, immature DCs scale bar = 500 μm, DCs scale bar = 500 μm. (B) DC marker expression via CD11b and CD141 in THP-1, ImDC, and mDC. (C) THP-1 and THP-1 cells were treated with 200 ng/ml IL-4, 100 ng/ml GM-CSF, 20 ng/ml TNFα, and 200 ng/ml Ionomycin for 72 hours and were incubated with IGF1 for 10 minutes before harvest, after which cell extracts were prepared. Proteins were separated through SDS-PAGE, followed by electrophoretic transfer and incubation with antibodies against tIGF1R, pIGF1R, tAKT, and pAKT. (D) Scanning densitometry analysis of tIGF1R (blue bars) and pIGF1R (red bars) levels in THP-1 and DCs. A value of 100% was given to the expression level of untreated THP-1 cells. Levels of tubulin were measured as a loading control. * P < .05. Bars represent SEM values. (E) Scanning densitometry analysis of pAKT levels in THP-1 and DCs. A value of 100% was given to the expression level of untreated THP-1 cells. Levels of tubulin were measured as a loading control.* P < .05. Bars represent SEM values. The graphs represent average of three independent experiments. (F) Expression of tIGF1R and pIGF1R with (green) or without (blue) IGF1 treatment in THP-1 cells.

Article Snippet: Cells were then washed and incubated with primary antibodies total IGF1R (tIGF1R) (sc-81,167, Santa Cruz Biotechnology, Inc.) and phospho IGF1R (pIGF1R) (Y1135/1136, Cell Signaling) for 1 hour at RT.

Techniques: Marker, Expressing, Incubation, SDS Page, Control

Journal: Translational Oncology

Article Title: IGF1R Axis Inhibition Restores Dendritic Cell Antitumor Response in Ovarian Cancer

doi: 10.1016/j.tranon.2020.100790

Figure Lengend Snippet: NVP-AEW541–treated DCs decrease ovarian cancer cell migration. (A) THP-1 myeloid cells were treated with 5 μM of NVP-AEW541 for 1, 24, and 48 hours. Whole-cell lysates were resolved on SDS-PAGE and immunoblotted with tIGF1R and pIGF1R antibodies. Level of tubulin was used as a loading control. Cell migration was detected by wound scratch assay. (B, left panel) Human leukemic THP-1 cells were differentiated to DCs and treated with 5 μM of NVP-AEW541, after which they were co-cultured with SKOV3; scratch was applied 24 hours after cell merge. The growth of EOC cells into the scratch zone is demonstrated here at time 0, 24,and 48 hours after scratch; scale bar = 500 μm. (B, right panel) Scanning densitometry analysis of % migration in co-culture of SKOV3 with DCs. A value of 100% was given to % migration in untreated DCs at 0 hour. (C, left panel) THP-1 cells were differentiated to DCs and treated with 5 μM of NVP-AEW541, after which they were co-cultured with ES2; scale bar = 500 μm. (C, right panel) Scanning densitometry analysis of % migration in co-culture of ES2 with DCs. A value of 100% was given to the expression level of untreated DCs at 0 hour. The graphs represent average % migration of three independent experiments of ES2 and SKOV3 cells. Images were captured at a magnification of ×4. * P < .05. Bars represent SEM value.

Article Snippet: Cells were then washed and incubated with primary antibodies total IGF1R (tIGF1R) (sc-81,167, Santa Cruz Biotechnology, Inc.) and phospho IGF1R (pIGF1R) (Y1135/1136, Cell Signaling) for 1 hour at RT.

Techniques: Migration, SDS Page, Control, Wound Healing Assay, Cell Culture, Co-Culture Assay, Expressing

Journal: Translational Oncology

Article Title: IGF1R Axis Inhibition Restores Dendritic Cell Antitumor Response in Ovarian Cancer

doi: 10.1016/j.tranon.2020.100790

Figure Lengend Snippet: Protein Expression (Intensity) Rates in EOC Tissue

Article Snippet: Cells were then washed and incubated with primary antibodies total IGF1R (tIGF1R) (sc-81,167, Santa Cruz Biotechnology, Inc.) and phospho IGF1R (pIGF1R) (Y1135/1136, Cell Signaling) for 1 hour at RT.

Techniques: Expressing, Staining

Journal: Translational Oncology

Article Title: IGF1R Axis Inhibition Restores Dendritic Cell Antitumor Response in Ovarian Cancer

doi: 10.1016/j.tranon.2020.100790

Figure Lengend Snippet: Relation Between Intensity (Protein Expression) and Clinicopathological Parameters of EOC

Article Snippet: Cells were then washed and incubated with primary antibodies total IGF1R (tIGF1R) (sc-81,167, Santa Cruz Biotechnology, Inc.) and phospho IGF1R (pIGF1R) (Y1135/1136, Cell Signaling) for 1 hour at RT.

Techniques: Expressing

Journal: Translational Oncology

Article Title: IGF1R Axis Inhibition Restores Dendritic Cell Antitumor Response in Ovarian Cancer

doi: 10.1016/j.tranon.2020.100790

Figure Lengend Snippet: Correlation Between the Mean Extensity of IGF1R, p53, Ki67, and Histological Grade

Article Snippet: Cells were then washed and incubated with primary antibodies total IGF1R (tIGF1R) (sc-81,167, Santa Cruz Biotechnology, Inc.) and phospho IGF1R (pIGF1R) (Y1135/1136, Cell Signaling) for 1 hour at RT.

Techniques:

Journal: Translational Oncology

Article Title: IGF1R Axis Inhibition Restores Dendritic Cell Antitumor Response in Ovarian Cancer

doi: 10.1016/j.tranon.2020.100790

Figure Lengend Snippet: Correlation coefficient between each individual values of IGF1R expression and CD1c in EOC patients ( P ˂.05).

Article Snippet: Cells were then washed and incubated with primary antibodies total IGF1R (tIGF1R) (sc-81,167, Santa Cruz Biotechnology, Inc.) and phospho IGF1R (pIGF1R) (Y1135/1136, Cell Signaling) for 1 hour at RT.

Techniques: Expressing

Journal: Theranostics

Article Title: Development of the phenylpyrazolo[3,4- d ]pyrimidine-based, insulin-like growth factor receptor/Src/AXL-targeting small molecule kinase inhibitor

doi: 10.7150/thno.48865

Figure Lengend Snippet: Synthesis of LL6 and determination of its cellular targets. ( A ) Superposed X-ray structures of AXL (magenta, PDB: 5U6B) and Src (yellow, PDB: 2SRC). Close-up views of overlaid binding pockets in cartoon (top) and surface (down) models are presented with the ligands shown in stick. ( B ) (i) Docking pose of PP-5 in the binding pocket of AXL. A close-up view of the binding orientation of PP-5 is shown in the box. See for further details. (ii) Suggested hydrogen bonding interactions of the critical binding site residues of AXL with PP-5 ; dashed green line and light green line represent conventional hydrogen bonds and carbon hydrogen bonds, respectively. (iii) Residues with hydrophobic interactions with PP-5 in the binding site of AXL. ( C ) Synthesis of compounds 3a-c, 4a-l, and 5a-h . Reagents and conditions: a) 6f , Cs 2 CO 3 , DMF, 80ºC, 3 h, 36-48%; b) 2-bromoethanol, Cs 2 CO 3 , DMF, 80ºC, 3 h, 50-63%; c) MsCl, pyridine, rt, 5 h, 45-76%; d) NaN 3 , DMF, 60°C, 8 h, 80-88%; e) 6a-e , CuSO 4 ·5H 2 O, NaAsc, tBuOH/H 2 O/DMF, 80ºC, 3 h, 39-80%. See Table for structural details. ( D ) Human kinome profile screened for LL6 at 10 µM. Red circles denote > 65% inhibition with a S-score of 0.12. The image was generated using TREE spot ™ analysis from KINOME scan (DiscoveRx). ( E ) Binding titrations for IGF-1R, Src, KIT, IR, AXL, and CSF1R were performed by competition binding assays from DiscoveRx. a Dose dependent inhibition curve was not obtained with these three proteins (IR, KIT, and CSF1R).

Article Snippet: Primary antibodies against IGF-1R and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Binding Assay, Inhibition, Generated

Journal: Theranostics

Article Title: Development of the phenylpyrazolo[3,4- d ]pyrimidine-based, insulin-like growth factor receptor/Src/AXL-targeting small molecule kinase inhibitor

doi: 10.7150/thno.48865

Figure Lengend Snippet: Effects of LL6 on the viability, colony formation, and migration of several NSCLC cells. ( A, D, E-G ) Dose-dependent inhibitory effects of LL6 on the viability of naïve ( A ), erlotinib-resistant ( D ), and chemoresistant ( E ) NSCLC cells and NSCLC colony formation grown in anchorage-dependent ( F ) and anchorage-independent ( G ) culture conditions evaluated using the MTT assay ( A, D, E ), anchorage-dependent colony formation assay ( F ), and soft agar colony formation assay ( G ), respectively. For cell viability assay, cells were treated with various concentrations of LL6 diluted in 10% FBS- ( A, E ) or 5% FBS ( D )-containing media for three ( A, E ) or two ( D ) days. ( B, C ) Western blot analysis showing basal expression of total and phosphorylated forms of AXL, IGF-1R, and Src in various NSCLC cell lines ( B ), PC9 ( C ), and erlotinib-resistant PC9 (PC9/ER) cells ( C ). ( H ) The effect of LL6 on the migration of NSCLC cells evaluated using Transwell migration assay. ( I ) The effect of LL6 on the viability of NSCLC cells under the same experimental conditions used for cell migration determined using the MTT assay. Bars represent mean ± SD. P < 0.05, ** P < 0.01 and *** P < 0.001, as determined using the two-tailed Student's t -test compared with the vehicle-treated control.

Article Snippet: Primary antibodies against IGF-1R and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Migration, MTT Assay, Colony Assay, Soft Agar Assay, Viability Assay, Western Blot, Expressing, Transwell Migration Assay, Two Tailed Test, Control

Journal: Theranostics

Article Title: Development of the phenylpyrazolo[3,4- d ]pyrimidine-based, insulin-like growth factor receptor/Src/AXL-targeting small molecule kinase inhibitor

doi: 10.7150/thno.48865

Figure Lengend Snippet: Improved efficacy of LL6 compared with the concurrent blockade of IGF-1R, Src, and AXL by treatment with a combination of linsitinib, dasatinib, and bemcentinib. ( A ) Inhibition of the phosphorylation of AXL, IGF-1R, and Src by treatment with LL6 was determined using Western blot analysis. ( B ) Inhibitory effects of LL6 on the phosphorylation of AXL, Src, and IGF-1R were determined using immunofluorescence analysis. ( C-E ) Inhibitory effects of LL6 (2.5 μM) on the viability ( C ), anchorage-dependent colony formation ( D ), and migration ( E ) evaluated in comparison with the treatment with a combination of linsitinib (L, 2 µM), dasatinib (D, 50 nM), or bemcentinib (B, 100 nM) (LD or LDB). ( F-H ) Proapoptotic effects of LL6 (2.5 µM), as determined by induction of PARP cleavage ( F ), chromatin condensation ( G ), and accumulation of the cell population in the sub-G1 phase ( H ), evaluated in comparison with the treatment with a combination of linsitinib (L, 2 µM), dasatinib (D, 50 nM), or bemcentinib (B, 100 nM) (LD or LDB). Bars represent mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, as determined using the two-tailed Student's t -test compared with the vehicle-treated control (A, B) and one-way ANOVA with Dunette's post-hot test (C-H). Scale bar: 20 µm.

Article Snippet: Primary antibodies against IGF-1R and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Immunofluorescence, Migration, Comparison, Two Tailed Test, Control

Journal: Theranostics

Article Title: Development of the phenylpyrazolo[3,4- d ]pyrimidine-based, insulin-like growth factor receptor/Src/AXL-targeting small molecule kinase inhibitor

doi: 10.7150/thno.48865

Figure Lengend Snippet: Inhibitory effect of LL6 on Kras G12D/+ -driven murine lung tumorigenesis. ( A ) Representative images showing inhibition of lung tumor formation in the lungs of LL6 (80 mg/kg)-treated mice. Left . Representative photographs of the lungs from vehicle- or LL6-treated mice. Right . Representative photographs of H&E-stained lung sections derived from vehicle- or LL6-treated mice. ( B-D ) Microscopic analysis of the H&E-stained lung sections for lung tumor multiplicity ( B ), tumor volume ( C ), and tumor burden ( D ). ( E ) Changes in body weight of vehicle- or LL6-treated mice. ( F ) The tumoral expression of phosphorylated AXL, Src, and IGF-1R (pAXL, pSrc, and pIGF-1R) in the lungs of vehicle- or LL6-treated mice was analyzed using immunohistochemistry. Bottom . Quantification of the positive cells for each marker per field of view. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001, as determined using the two-tailed Student's t -test compared with the vehicle-treated control. Scale bars: 50 µm.

Article Snippet: Primary antibodies against IGF-1R and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Staining, Derivative Assay, Expressing, Immunohistochemistry, Marker, Two Tailed Test, Control

Journal: Theranostics

Article Title: Development of the phenylpyrazolo[3,4- d ]pyrimidine-based, insulin-like growth factor receptor/Src/AXL-targeting small molecule kinase inhibitor

doi: 10.7150/thno.48865

Figure Lengend Snippet: Inhibition of the growth of xenograft tumors and spontaneous lung metastasis of LLC allografts by administration of LL6 in vivo . ( A ) Inhibition of tumor growth (left) and tumor weight at endpoint (middle) by treatment with LL6 (80 mg/kg) with negligible changes in body weight (right). ( B ) Suppression of the activation of IGF-1R and Src in tumors from mice treated with LL6. Right . Quantification of the level of phosphorylated IGF-1R or Src expression versus corresponding total protein expression by densitometric analysis using ImageJ software. ( C ) Significant reduction in metastatic tumor formation in the lungs after treatment with LL6 (80 mg/kg). Bioluminescence imaging shows a decrease in tumor formation in the lungs of LL6-treated mice. Bars represent mean ± SD. P < 0.05 and P < 0.01, as determined using the two-tailed Student's t -test compared with the vehicle-treated control.

Article Snippet: Primary antibodies against IGF-1R and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, In Vivo, Activation Assay, Expressing, Software, Imaging, Two Tailed Test, Control

Journal: Oncology Letters

Article Title: Receptor tyrosine kinase expression in high-grade gliomas before and after chemoradiotherapy

doi: 10.3892/ol.2019.11017

Figure Lengend Snippet: Significantly changed receptor tyrosine kinases between initial and recurrent gliomas.

Article Snippet: 5% skim milk was used to block the membrane at room temperature for 1 h. Primary rabbit polyclonal antibodies against IGF1R (ProteinTech Group, Inc.; cat. no. 10297-1-AP; dilution, 1:200) were used at 4°C overnight.

Techniques:

Journal: Oncology Letters

Article Title: Receptor tyrosine kinase expression in high-grade gliomas before and after chemoradiotherapy

doi: 10.3892/ol.2019.11017

Figure Lengend Snippet: IGFR was activated by TMZ. (A) Western blot analysis revealed that IGF1R expression was increased in glioma cells after treatment with TMZ. Expression levels was normalized by the level of GAPDH and expressed as fold-changes of those in parental cells. The data are presented as mean ± SD. ***P<0.001 as indicated. (B) Data analysis of 135 initial and 47 recurrent high-grade glioma cases revealed that IGFR signaling was highly activated in recurrent gliomas. TMZ, temozolomide; IGF1R, insulin-like growth factor 1 receptor.

Article Snippet: 5% skim milk was used to block the membrane at room temperature for 1 h. Primary rabbit polyclonal antibodies against IGF1R (ProteinTech Group, Inc.; cat. no. 10297-1-AP; dilution, 1:200) were used at 4°C overnight.

Techniques: Western Blot, Expressing